The present invention relates generally to detection of nucleic acid sequences, and, more particularly, to the selective incorporation of fluorescent markers to detect nucleic acid sequences.
The rapid and efficient detection of specific nucleic acid sequences in biological samples plays a central role in a variety of fields, including molecular biology, biotechnology, immunology, medical diagnosis, forensic analysis, and quality control of food products. One of the most commonly used techniques for the detection of specific nucleic acid sequences is the Southern blot. This is a hybridization technique in which the fragments to be interrogated have been size-separated by gel electrophoresis and transferred from the gel to a nylon nitrocellulose filter. A radioactive probe is then added to the filter so that hybridization takes place. After washing away the excess probe, the band containing the target nucleic acid is detected by exposing an x-ray film to the filter.
Despite its popularity, Southern blotting suffers from some limitations: it involves a series of manually intensive procedures that cannot be run unattended and cannot be readily automated. The process for separating the fragments by gel electrophoresis and subsequently detecting the bands by autoradiography are time-consuming tasks that are susceptible to poor quantitative accuracy and poor reproducibility.
The use of radioactive probes brings up a set of safety and environmental concerns. The lack of adequate sensitivity is another limitation, which has been partially addressed by the development of the polymerase chain reaction (PCR) and related target amplification methods. The PCR consists of selectively amplifying a target DNA sequence in a sample. Amplification products are usually detected by dyes that stain nucleic acids or by hybridization with sequence-specific probes. Amplification methods, however, may introduce ambiguities resulting from contamination or from variability in amplification efficiency. Therefore, there is a need for robust analytical methods that provide accurate quantitation and molecular weight estimates for target DNA or RNA segments.
Various objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention includes a method for identifying a target DNA or RNA sequence. A primer having a 3xe2x80x2-hydroxyl group at one end and having a sequence of nucleotides sufficiently homologous to hybridize with an identifying sequence of nucleotides in the target DNA or RNA is selected. The primer is hybridized to the identifying sequence of nucleotides and a reporter molecule is synthesized on the target sequence by extending the primer by progressively binding nucleotides to the primer that are complementary to the corresponding nucleotides of the DNA or RNA sequence, where the complementary nucleotides include nucleotides labeled with a fluorophore. Fluorescence emitted by fluorophores on individual reporter molecules is detected to identify the target DNA or RNA sequence.